A Molecular Model of Phosphorylation-Based Activation and Potentiation of Tarantula Muscle Thick Filaments

Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020A, Venezuela.
Journal of Molecular Biology (Impact Factor: 3.96). 09/2011; 414(1):44-61. DOI: 10.1016/j.jmb.2011.09.017
Source: PubMed

ABSTRACT Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ~50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca(2+)-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads ("free" and "blocked") are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states ("swaying" heads). These heads would be available for immediate actin interaction upon Ca(2)(+) activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca(2+) on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.
    Biophysical Journal 10/2014; 107(7):1637-1646. DOI:10.1016/j.bpj.2014.07.075 · 3.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A conditioning maximal voluntary muscle action (MVC) has been shown to induce post-activation potentiation, i.e. improved contractile muscle properties, when muscles are contracted isometrically. It is still uncertain how the contractile properties are affected during ongoing muscle length changes. The purpose of this study was to investigate the effects of a 6 s conditioning MVC on twitch properties of the plantar flexors during ongoing muscle length changes. Peak twitch, rate of torque development (RTD) and relaxation (RTR), rising time and half relaxation time (HRT) were measured from supramaximal twitches evoked in the plantar flexors of 11 highly trained athletes. Twitches were evoked prior to a 6 s MVC and subsequently on 8 different occasions during a 10-minute recovery, for five different modes: fast lengthening, slow lengthening, isometric, fast shortening and slow shortening of the plantar flexors. The magnitude and duration of effects from the conditioning MVC were significantly different between modes. Peak twitch, RTD and RTR significantly increased for all modes but more so for twitches evoked during fast and slow shortening as compared to lengthening. Rising time was reduced in the lengthening modes, but slightly prolonged in the shortening modes. HRT was significantly reduced for all modes except fast lengthening. The findings show that the effects of a conditioning MVC on twitch contractile properties are dependent on direction and velocity of ongoing muscle length changes. This may imply that functional enhancements from a conditioning MVC might be expected to be greatest for concentric muscle actions, but are still present in isometric and eccentric parts of a movement.
    Medicine and science in sports and exercise 07/2014; 46(7). DOI:10.1249/MSS.0000000000000245 · 4.46 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The overall conformations of regulated myosins or heavy meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the "off" state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the "on" state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on heavy meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca(2+) the radius of gyration increases. Differences in the squid "on" and "off" states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca(2+)-free squid heavy meromyosin that is compact, but which becomes extended when Ca(2+) is bound. Further, the scattering profile derived from the current model of tarantula heavy meromyosin in the "off" state is in excellent agreement with the measured "off" state scattering profile for squid heavy meromyosin. The previous and current studies together provide significant evidence that regulated myosin's compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.
    PLoS ONE 12/2013; 8(12):e81994. DOI:10.1371/journal.pone.0081994 · 3.53 Impact Factor